Molecular analysis of F. nucleatum interspecies interactions in biofilms

描述(申请人提供)：口腔中有一个复杂的微生物生物膜群落，能够介导急性和慢性多菌感染，包括牙周病，这仍然是全球最常见的牙齿脱落原因。特定的物种间相互作用是建立这些群落的关键因素，而核梭杆菌(Fn)已被确定为中心的“桥梁”有机体，使许多口腔细菌物种(包括“早期”和“晚期”定殖者)能够排列成组织良好的群落，以形成生物膜和致病。通过与浮游细胞的体外共聚集研究，已经清楚地证明了FN与多种口腔物种的黏附能力，但只有有限的研究探索这种能力如何在多物种微生物生物膜中表现出来。更重要的是，缺乏有效的遗传操作工具和明确的分子靶标，阻碍了对FN介导的这些迷人的物种间相互作用背后的分子机制的研究。我们最近首次获得了梭状细菌粘附素(RADD)的基因特征，这种粘附素可以介导物种间与早期口腔定殖者的粘连。在我们的初步研究中，我们发现依赖于辐射的与其伙伴血链球菌(SS)的结合可以刺激FN明显的表型变化和差异基因表达。基于这些发现，我们提出了我们的工作假说，即FN通过RADD及其相关的Rad蛋白与早期定植的伙伴物种相互作用，这引发了FN的显著形态和生理变化，这对生物膜的形成至关重要。为了验证这一假设，我们以血链球菌(SS)为模型，详细研究了RADD在FN介导的种间相互作用中的作用。这些方法将包括表征生物膜的行为、口腔群落的整合、基因表达模式以及鉴定SS中参与OMP介导的与FN相互作用的细胞成分。这一全面的方法将在分子水平上为生物膜形成中涉及的事件提供详细的知识，包括物理细胞-细胞接触和信号传递。这项研究将为改进对一种广泛存在的人类传染病的治疗干预提供新的靶点，并为未来研究晚期定植伙伴物种或动物模型的FN奠定基础。

DESCRIPTION (provided by applicant): The oral cavity harbors a complex microbial biofilm community capable of mediating acute and chronic polymicrobial infections including periodontal diseases which are still the most common reason for tooth loss worldwide. Specific interspecies interactions are key factors in constructing these communities and Fusobacterium nucleatum (Fn) has been established as the central "bridging" organism that enables arrangement of numerous oral bacterial species (both "early" and "late" colonizers) into well organized communities for biofilm formation and pathogenesis. The ability of Fn to adhere to a large variety of oral species has been clearly demonstrated through elaborate in vitro co-aggregation studies with planktonic cells, yet only limited studies have explored how this ability manifests in multispecies microbial biofilms. More importantly, lack of effective genetic manipulation tools and clear molecular targets has hampered investigation of the molecular mechanisms behind these Fn-mediated fascinating interspecies interactions. We recently achieved the first genetic characterization of a fusobacterial adhesin (RadD) that mediates interspecies adhesion with early oral colonizers. In our preliminary studies, we demonstarted that RadD- dependent binding to its partner species Streptococcus sanguinis (Ss) stimulates distinct phenotypic changes and differential gene expression in Fn. Based on these findings, we developed our working hypothesis that "Fn interacts via RadD and associated Rad-proteins with early colonizing partner species, which triggers significant morphological and physiological changes in Fn important for biofilm formation". To test this hypothesis, we propose a detailed examination of the role of RadD in Fn-mediated interspecies interactions using Streptococcus sanguinis (Ss) as model early colonizing partner species. Approaches will include characterization of biofilm behavior, oral community integration, gene expression patterns and identification of cellular components in Ss involved in the OMP-mediated interaction with Fn. This comprehensive approach will provide detailed knowledge at the molecular level for the events involved in biofilm formation including physical cell-cell contact and signaling. This research will provide novel targets for improved therapeutic intervention of a widespread human infectious disease and serve as a basis for future studies of Fn with late colonizing partner species or animal models.